Project Summary Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease without cure. Most ALS are sporadic cases without identified genetic causes. However, the spinal cord and muscle autopsy/biopsy samples from both sporadic and familial ALS patients all show remarkable defects in morphology and biochemical properties of mitochondria. This indicates abnormal mitochondria as a common player in neuromuscular degeneration despite the etiology. Our research using the ALS mouse models (G93A), over the last 12 years, establishes a concept that mitochondrial dysfunction in skeletal muscle is part of the pathogenesis of ALS. Muscle appears to be a primary target of ALS mutation, in addition to being victim of neuronal withdrawal, because mitochondrial defects in muscle feedback to neuromuscular junction (NMJ) remodeling in ALS. Thus, restoration of mitochondrial function is a logical approach to alleviate the systemic symptom of ALS through fixing a common pathology. We made a novel discocery that ALS progression includes a leaky gut with an imbalanced microbiome (dysbiosis) in G93A mice. This gut defects occurs before the onset of ALS neuromuscular symptoms, suggesting that gut defects may play a role in ALS progression. We reported that the colon of G93A mice contained less butyrate-producing bacteria, and the dietary butyrate supplementation alleviated gut defects in G93A mice, improving their neuromuscular performance and extending their life span. Thus, our study brought a new concept that restoring gut homeostasis may provide an alternative means for improving neuromuscular function to treat ALS. Since the original submission, our collaboration with the Brotto Lab made several exciting new discoveries. We identified altered Lipidomics Profiles of ROS-related Bioactive Lipids (BLs) in muscle that were restored by one-month butyrate diet supplementation in G93A mice. Further, butyrate treatment directly enhanced muscle contractility. Our preliminary data also show that butyrate treatment improved mitochondrial function and its susceptibility to oxidative-stress induced damage in G93A muscle fibers. Our data suggest that butyrate could be an important mediator regulating the neuromuscular-gut integrative physiology. We hypothesize that integrative signaling between the neuromuscular system and gut contributes to the progressive loss of mitochondrial function in ALS, and restoration of butyrate- related microbiome has benefits in preserving mitochondrial function for treatment of ALS. The proposed study will address two fundamental questions: How do gut defects contribute to mitochondrial dysfunction of neuromuscular system in ALS (Aim 1)? Can neuromuscular-gut signaling be leveraged to improve mitochondrial function to slow ALS progression and/or improve the life quality of ALS patients (Aim 2)? While altered intestinal homeostasis and microbiome is linked to the human pathology of ALS, we anticipate that our study will bring novel concepts to the ALS research field. Knowledge gained from this study can have potential translational implications for developing new therapeutic strategies for combating ALS.